It is a goal in the nucleic acid probe art to detect various nucleic acid sequences in a biological sample, in which the sequences, as so-called target nucleic acid, are present, usually in small amounts relative to the wide variety of other nucleic acid species, including RNA, DNA or both. Thus, it is desirable to be able to detect the RNA encoding polypeptides that may be associated with diseases or pathological conditions, such as, for example, RNA of the genome of the human immunodeficiency virus. In addition to the detection of RNAs associated with such viruses, it is desirable to detect other RNAs characteristic of, for example, a disease or pathological condition, such as those transcribed from a defective gene, as in the case of hemophilia or sickle-cell anemia. Characteristically, the RNA associated with such a disease or condition is present, if at all, in very small amounts relative to total nucleic acid in a given biological sample, such as a sample of blood or other body fluid or a tissue sample of an individual to be tested.
The detection of such an RNA species requires such specificity and sensitivity that, if the RNA is present, it is detectable and measurable from amongst the wide variety of other nucleic acid species with which it is associated in a sample. Some of these non-target nucleic acid species may bear close homology, at least in isolated segments, to the target RNA. Further, as noted above, these target RNA species are very often found only in very minute amounts in the biological sample being tested. Yet, for proper diagnosis of the underlying disease state, it is essential that even small amounts of a target RNA be detectable if present.
Several approaches have been advanced for accomplishing reliable detection, in a biological sample, of a target nucleic acid present, if at all, in only a small amount and as only a minute fraction of total nucleic acid. In one approach, the amount of nucleic acid in the sample is not altered. Instead, a reporter system is employed whereby a large number of readily detectable molecules is produced for each molecule of target nucleic acid in the sample and the presence or quantity of the detectable molecules is measured. Such a reporter system is a signal-generating system associated with the target nucleic acid and produces a detectable signal representative of the number of molecules of target nucleic acid in a sample.
In another, fundamentally different, approach a target nucleic acid segment that is part of the target nucleic acid but not other nucleic acids in biological samples to be assayed, or the complement of such a target segment (i.e., the segment with the same size as, but sequence complementary to, that of the target segment), or both the target segment and its complement are selectively increased in copy number. That is, in a sample, the copy number of the target nucleic acid segment or its complement, or the copy numbers of both the target segment and its complement, are increased to a greater extent than the copy number of any other nucleic acid segment. This selective increase in copy number of a nucleic acid segment is referred to in the art as "amplification" of the segment. Once a target segment (also referred to herein, and in the art, as a "target sequence") or the complement of a target segment is amplified to a sufficient extent, it can be detected reliably by any of many techniques that have been developed in the nucleic acid probe art for the detection of nucleic acid segments, including techniques which involve the first approach, described above, that entails production of many readily detectable molecules for each molecule of target nucleic acid.
One method that has been developed for the amplification of a target segment is the so-called "polymerase chain reaction" ("PCR") method. This technique was reported by Saiki et al., Science 230, 1350 (1985) and Mullis et al., European Patent Application Publication Nos. 200362 and 201184 (See also U.S. Pat. Nos. 4,683,195 and 4,683,202), and particularly entails repeated cycles of (1) hybridizing to the 3'-end of a target nucleic acid sequence a first primer and to the 3'-end of the sequence complementary to the target sequence a second primer, (2) extending the primers with a polymerase, and (3) rendering single stranded the duplexes resulting from the chain extension reaction. This PCR procedure results in amplification of the target sequence and its complement exponentially with the number of cycles (i.e., as in a chain reaction).
Certain RNAs are known to be susceptible to autocatalytic replication by RNA-dependent RNA polymerases. Among the polymerases which are capable of such autocatalytic replication are bacterial phage RNA-dependent RNA polymerase such as Q.beta. replicase. Autocatalytically replicatable RNAs are said herein to have "template sequences" which means they have sequences that make them templates for replication by the polymerase. In the process, the RNA made from a template RNA (i.e., an RNA with a "template sequence") in the reaction catalyzed by the replicase is also replicatable RNA (i.e., also has a "template sequence"). Thus, in autocatalytic replication, the amount of replicatable RNA can increase exponentially. See Miele et al., J. Molecular Biology 171, 281 (1983).
Until recently it has not been appreciated that autocatalytic replication could be employed to provide convenient, broadly applicable, highly sensitive reporter systems for analyses of biological samples for the presence of particular nucleic acid sequences. A system in which probe for a target sequence is linked to an RNA capable of being replicated by Q.beta. replicase is described in U.S. Pat. No. 4,957,858, and by Chu et al., Nucleic Acids Research 14, 5591 (1986), the disclosures of which are expressly incorporated herein by reference. Thus, the invention described in U.S. Pat. No. 4,957,858 combines the art of replication of RNA with the art of nucleic acid hybridization probes by employing, as a signal producing moiety in a reporter system for detection of a target molecule, a replicatable RNA which is associated (through a probe, such as an oligonucleotide probe) with a target segment. A disadvantage of using such a nucleic acid hybridization assay having, as a reporter system, a replicatable RNA is that non-specific binding of a probe-replicatable RNA moiety to, for example, the walls of an assay tube can result in high levels of noise (i.e., low signal to noise ratios) and/or false positive readings.
It would be desirable to be able to combine the advantages of target nucleic acid amplification systems, where the target nucleic acid sequence itself is amplified, with the advantages of nucleic acid hybridization assays employing replicatable RNAs as a reporter system, while eliminating disadvantages of the former (such as the need for repeated thermal cycling steps) as well as disadvantages of the latter, such as nonspecific binding of a reporter moiety which is capable of autocatalytic replication even in the absence of specific binding to a target sequence.
It is an object of the present invention to provide compositions and methods for detection of RNA target segments using a process that, in assuring the presence of the target sequence in the amplified product, avoids or at least substantially reduces the presence of low signal-to-noise ratios and false positives. It is a further object of the present invention to provide target RNA amplification compositions which utilize portions of autocatalytically replicatable RNAs and which result in the production of recombinant replicatable RNAs, such that autocatalytic replication of the recombinant RNAs results in amplification of a target nucleic acid of interest (i.e., a segment of the target RNA) which is foreign to the replicatable RNA.
It is thus an overall object of the present invention to meet the goals enumerated by the art and to provide selective means to meet disadvantages and problems encountered in the prior art. The methods and compositions of the present invention result in the production of recombinant, replicatable RNA molecules that are produced only where the necessary target nucleic acid sequence is present in the sample tested.